Human myeloproliferative neoplasms:molecular mechanisms, diagnosis and

classification

Tony GreenCambridge Institute for Medical Research

University of Cambridge and Addenbrookes Hospital

1. Normal mammaryepithelial development

2. First driver mutation

3. Further driver mutationsand clonal expansions

4. Appearance of mostrecent common ancestor

5. Completion of lasttotal selective sweep

6. Final rate-limitingdriver mutation

7. Diagnosis

Nik-Zainal et al Cell 2012

Myeloproliferative neoplasms

ProgenitorsBlood stem cell

Red cells

Platelets• Arise in blood stem cell

• Increased production of mature cells

• Window on earliest stage of tumorigenesis

• Tractable – accessible tissue, chronic diseases, clonal analysis

The BCR-ABL negative myeloproliferative neoplasms

Polycythaemia Vera (PV)

Essential thrombocythaemia

(ET)

Acute myeloid leukaemia

JAK2 V617F mutation

60%

95%

The JAK2 V617F mutation

James et al Nature 2005; Baxter et al Lancet 2005Levine et al Cancer Cell 2005; Kralovics et al NEJM 2005Scott et al NEJM 2007; Bercovich et al Lancet et al 2008

A T G T N T C T A T G T G T C T

Grans T cells

A T G T T T C T

Homozygous Mitoticrecombination

FERM SH2 JH2 Kinase

V617FExon 12

neg

JAK2

V617F mutation

Exon 16 mutations

Exon 12 mutations

“It all starts to make sense”

PI3K

pSTAT5

MAPK

P P

P P

PI3K

pSTAT5

MAPK

PP

P P

EPORTPOR

JAK2

Diagnosis

Appearance of mostrecent common ancestor

Rapid direct clinical impact

Identification of JAK2

mutation

PT-1 trials

Specialist MPN clinic

Recognition of

new disease

subtypes

Molecular testing in regional

diagnostic service

Therapeutic

JAK2 inhibitors

Harrison et al NEJM 2005

Baxter et al Lancet 2005

Campbell et al Lancet 2005

Scott et al Blood 2006

Campbell et al Blood 2006a

Campbell and Green NEJM 2006

Campbell et al Blood 2006b

Scott et al NEJM 2007

Wilkins et al Blood 2008

Beer et al Blood 2008

Zhao et al NEJM 2008

Campbell et al JCO 2009

Beer et al Blood 2010

Chen et al Cancer Cell 2010

Godfrey et al Blood 2012

Selected Green lab translational papers since 2005

2005 2010

A 1 Red cell mass >25% above predicted or Hct >60% male,

>56% female

2 No cause for 2o erythrocytosis

3 Palpable splenomegaly

4 Clonality marker

B 1 Platelet count >400 x109/l

2 Neutrophils >10 x109/L (>12.5 in smokers)

3 Splenomegaly on imaging

4 Endogenous erythroid colonies or reduced serum epo

A1 + A2 + any other A establishes diagnosis

A1 + A2 + two of B establish diagnosis

Old criteria for diagnosis of PV

Diagnostic criteria 20012

1 Raised Hb

2 JAK2 mutation

3 No cause for 20 erythrocytosis - normal art O2

- serum epo not high

McMullin et al BCSH guidelines BJH 2005, 2007

Idiopathic erythrocytosis or PV?

Scott et al NEJM 2007

FERM SH2 JH2 Kinase

V617FExon 12wt ATGGTGTTTCACAAAATCAGAAAT M V F H K I R N

mut ATGGTGTTTCAATTATTAATCAGAAAT M V F Q LQ L I R N

granulocytes

erythroid colony

PV variant with JAK2 exon 12 mutations

Glycophorin A

Scott et al NEJM 2007; Percy et al Haematologica 2007; Boyd et al BJH 2010

H&E

Multiple mutations

Isolated erythrocytosis

High Resolution Melt Analysis

granulocytes

erythroid colony

Can be low level in blood

Diagnosis of ET: JAK2 or MPL mutation-positive

WHO 2008

Sustained platelet count >450

Acquired mutation (JAK2 or MPL)

No other myeloid malignancy

Typical bone marrow appearances

(reticulin ≤ grade 2)

BCSH guidelines 2009

Sustained platelet count >450

Acquired mutation (JAK2 or MPL)

No other myeloid malignancy

BUT in absence of mutation still need to exclude reactive causes

Distinguishing different MPNs

ET PV PMF

Beer et al Blood 2011

diagnose and

Distinguishing different MPNs

ET PV PMF

Beer et al Blood 2011

diagnose and

ET is heterogeneous

Key issue – can Cologne/WHO histological criteria identify distinct disease entities

WHO book is like a country – with good aspects…

….. but some aspects less good

….. but some aspects less good

Three questions

• Is prefibrotic PMF really distinct

• Can WHO criteria be applied reproducibly

• Is prefibrotic MF a useful concept

ET

Campbell et al Lancet 2005

Scott et al Blood 2006

Distinguishing ET from PMFRole of histology

“True ET” “Prefibrotic PMF”

Wilkins et al Blood 2008 Campbell et al JCO 2009

?

• 3 experienced haematopathologists, large prospective cohort (PT1 study) , blinded analysis of

WHO diagnosis and 16 morphological criteria

Even experienced haematopathologists don’t agree on what to call

“true ET” & “prefibrotic PMF”

Two alternative explanations:

- ‘Prefibrotic PMF’ not a distinct entity

- ‘Prefibrotic PMF’ exists but special training required

- even if true, questionable utility of criteria

the application of which is so difficult even

for highly experienced pathologists

Conclusions

Utility of prefibrotic PMF criteria (WHO 2008)

Thiele et al Blood 2011

Barbui, Thiele et al JCO 2011

Brousseau et al Histopathology 2010 “Distinction between ET and prefibrotic

PMF is of questionable clinical relevance”

Buhr, Kreipe et al Haematologica 2012 > 50% no agreement or unclassifiable -

“WHO criteria for discriminating ET from prefibrotic PMF are poorly to only moderately reproducible”

YES:

NO:

Concordance 73-88%

Barbui, Thiele et al JCO 2011

• Limitation of consensus approach to histology

• MF progression at 15 yrs: 10% vs 17% true ET vs prefib

MF • Leuk progression at 15 yrs 2% vs 11% - but no mention of therapy

• Even if real difference – prefibrotic PMF likely to represent later stage of same disease process

Three questions

• Is prefibrotic PMF really distinct

• Can WHO criteria be applied reproducibly

• Is prefibrotic PMF a useful concept

? later stage

disease

NO

Nomenclature of “prefibrotic PMF” is flawed

“True ET”

PV PMF

Prefibrotic PMF?

Nomenclature of “prefibrotic PMF” is flawed

ET PV PMF

Prefibrotic PMF?

Nomenclature of “prefibrotic PMF” is flawed

ET PV PMF

5-30%

Nomenclature of “prefibrotic PMF” is flawed

ET PV PMF

Prepolycythaemic PV?

Concept of “prefibrotic PMF” is also potentially dangerous

For individual patient management - inappropriate therapy (eg BMT) for low risk patients

For the MPN field - patient cohorts will not be comparable

Three questions

• Is prefibrotic PMF really distinct

• Can WHO criteria be applied reproducibly

• Is prefibrotic PMF a useful concept

? later stage disease

NO

FlawedDangerous

Thrombocytosis with isolated increased reticulin

Distinguishing ET from PMF

• Relatively common – 15-20%

• Unclassifiable under WHO

• Benign prognosisImplications – patient predisposed to robust fibrosis but lacks 2nd hits needed for evolution of clinical disease

Distinguishing ET from PMFPMF as presentation in accelerated phase

of pre-existing MPN

• PMF and MF transformation

indistinguishable

• PMF patients may have prior thrombocytosis

• PMF exhibits features of late stage disease - high clonal burden – more cytogenetic abnormalities - increased progression to AML

Campbell and Green NEJM 2006

Molecular classification of ET and PMF

Chronic phase

Heterogeneous

(mostly Pl)

Includes MF transformn

+ some PMF and atypical

CML

Accel phase

WBC retic dysplasia

Leuk phase

JAK2 MPL

Beer et al Blood 2011

Mutation load

Distinguishing different MPNs

ET PV PMF

Beer et al Blood 2011

diagnose and

JAK2-positive ET is forme fruste of PV

809 ET patients in PT-1 trial

Campbell et al Lancet 2005 Scott et al Blood 2006

Distinguishing ET from PV

Higher Hb and WBCIncreased e’poiesis and g’poiesisMore venous thrombosisMore transformation to PV

JAK2 mut neg JAK2 mut pos

Campbell et al Lancet 2005

Harrison et al NEJM 2005

Suppression of Epo in JAK2-pos ET

Haemoglobin (g/dL)

Seru

m E

po (

mU

/mL)

0

5

10

15

20

25

30

35

40

11 12 13 14 15 16

JAK2 WT

JAK2 V617F

p<0.0001

Distinguishing ET from PV

N

14 16 18 20 Hb

Normal ET PV

Inherent problem in using continuous variable (eg Hct or RCM) to make a binary distinction

Polycythaemia vera

Essential thrombocythaemia

Mitotic recombination

Rare Common

One mutation but two diseasesOne mutation but two diseases

Hypothesis – homozygosity for JAK2 mutation causes PV phenotype

JAK2JAK2V617FV617F knockin mouse knockin mouse – homozygosity – homozygosity causes phenotypic switch from ET to PVcauses phenotypic switch from ET to PV

Li et al Blood 2010 Juan Li, David Kent unpublished

Pla

tele

ts (

x1

0x

1033 )

/)/µµ

ll

Ha

em

ato

cri

t (%

)

WT Het Hom WT Het Hom

Haematocrit Platelets

Heterozygous

JAK2V617F

9p LOH

ET

PV

Higher number of homozygous colonies in PV patients compared to ET

- homozygous clone has selective advantage in PV but not ET

- recurrent acquisition of homozygosity

PV 80% ET 52%

Homozygous mutant BFU-EHomozygous mutant BFU-E present in many patients with ETpresent in many patients with ET

Godfrey et al Blood 2012

44 5 4

Mb from telomere

4.0 D9S2884.8 D9S18106.2 D9S1852

14.8D9S235

18.3D9S925

19.7D9S162

27.6 D9S161

36.4D9S1791

38.3D9S2148

30.9 D9S43

33.9 D9S1817

102.1 D9S176

Cen

Tel

JAK2

HeterozygousLOH

125 130 135 125 130 135 125 130 135

A CB170 180 170 180 170 180

Recurrent acquisition of 9p LOH in patient with PV

Number of colonies

A B C

• Recurrent acquisition of homozygosity - in 5/8 PV patients and 2/2 ET patients tested - resolution limited (2.3 to 14.2 Mb) so number of

distinct clones may be underestimate

• PV distinguished from ET by presence of dominant homozygous clone ~10 fold larger than minor clones

- ? additional lesions

• Multiple clones arise in HSPC compartment - persist over time - detectable in sorted CMPs

Summary

Heterozygous

JAK2V617F

Recurrent 9p LOH

ET

PV

IFNG up-regulated genes

NES: -2.156q val: <0.002

PV ET

PV

MUTWTET

pSTAT1

DAPI pSTAT1 Actin

Differential STAT1 signaling in heterozygous colonies from patients with ET and PV

Arrays Pathway

Key regulator

Chen et al Cancer Cell 2010

Polycythaemia vera

Essential thrombocythaemia

JAK2 V617F JAK2 V617F

STAT1 defect

JAK2 homozyg

Clonal expansion

• Unexpected complexity in early phase of “simple” malignancy

• Questions - how does clone expand given HSC defect - what drives recurrent mitotic recombination - what drives expansion of dominant homozygous

clone in PV - cause and effects of pSTAT1 defect in heterozygous

PV cells

• Exomes coming

Conclusions and questions

AcknowledgementsSanger Institute

Peter Campbell, Mike StrattonAndy Futreal, Elli Papaemmanuil

Cambridge UniversityAnne Ferguson-Smith

Carol Edwards

Nick CrossAmy Jones

Claire Harrison

Mary-Frances McMullin

Adam MeadSten-Eirik Jacobsen

Alessandro Vannucchi

Eva Hellstom

Ghulam Mufti

Jean Jacques Kiladjian

Green labMaria AhnAthar AzizPhilip BeerEdwin ChenJyoti Evans

Anna GodfreyTina Hamilton

David KentJuan Li

Steve LoughranCharlie Massie

June ParkDean Pask

Yvonne SilberRachel Sneade

Addenbrookes/BRCMike Scott

Joanna Baxter/Anthony Bench

MPD clinic, TRL team

NCRI MPN Study GroupPT-1 trial team

The myeloproliferative neoplasms

PrimaryMyelofibrosis

Polycythaemia Vera (PV)

Essential thrombo- cythaemia (ET)

Acute myeloid leukaemiaThrombosis

JAK2 mutations

FERM SH2 JH2 Kinase

exon 14

V617F

95% PV

50% ET

50% IMF

neg

exon 12 mutation

s

V617F-neg PV variant

exon 16 R683

Acute Lymphobla

stic Leukaemia

Difference between mutation pos ET and PV

JAK2Mutation

Normal Hb

Erythrocytosis

Depletediron stores

Genetic modifiers

Gender

Campbell et al Lancet 2005

Acquired mutations

Scott et al NEJM 2007

Nuclear JAK2 signaling directly regulates chromatin structure

JAK2 is a histone H3 kinase

H3Y41

H3Y54 H3Y99Dawson et al Nature

2009Collab with Kouzarides

lab

JAK2 signaling to chromatin mediates ES cell self-renewal

Griffiths et al Nat Cell Biol 2011

Collab with Gottgens lab

H3Y41

P H3Y41

P Nanog